缺氧诱导下人微血管内皮细胞的活化与腺苷水平的关系  

Relationship of human microvascular endothelial cell activation and adenosine level under the hypoxia

作　　者：艾李倩玉 黄婵娟[1] 陈琛[1] 林森[1] 叶剑[1] 

机构地区：[1]第三军医大学大坪医院眼科,重庆400042

出　　处：《中华实验眼科杂志》2017年第1期26-31,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：全军十二五医学发展计划项目（13CXZ051）

摘　　要：背景目前普遍认为视网膜局部缺氧状态是诱导视网膜新生血管形成的主要原因之一，其发病机制研究是国内外的研究热点。研究证实缺氧刺激下多种转录因子活性增加可促进微血管内皮细胞活化，导致新生血管形成，其中除血管内皮生长因子外，腺苷促血管生成作用也逐渐受到关注。目的研究缺氧诱导下微血管内皮细胞的活化过程与腺苷水平之间的关系，探讨腺苷微环境对血管生成的促进作用。方法对人微血管内皮细胞株（HMEC-1）进行体外培养，将培养的细胞分为常氧组和缺氧组，常氧组细胞置于5％CO2的细胞培养箱内培养，缺氧组细胞置于体积分数1％O1、94％N1和5％CO1的细胞培养箱内培养。采用CCK8法和EDU法检测各组细胞增生的吸光度（A值）和增生比例；采用Transwell小室法检测各组迁移细胞数与侵袭细胞数；采用Western blot法检测细胞中CD39和CD73蛋白的相对表达水平，采用免疫荧光法检测细胞中CD39和CD73的表达；采用高效液相仪检测各组细胞中的腺苷浓度。结果常氧组和缺氧组培养后12h细胞增生值（A值）分别为0．715±0．067和0．821±0．056，培养后24hA值分别为0．946±0．028和0．998±0．028，缺氧组12h组和24h组细胞增生值均高于常氧组，差异有统计学意义（t12h=3．805、t24h=3．222，均P〈0．01）；缺氧组细胞培养后24h细胞增生比例明显高于常氧组，差异均有统计学意义（t=-6．868，P〈O．01）。常氧组和缺氧组细胞培养24h迁移细胞数分别为185．3±10．594和300．7±22．853，侵袭细胞数分别为74．2±10．741和107．5±7．007，缺氧组迁移和侵袭细胞数均明显多于常氧组，差异均有统计学意义（t=-12．124、-6．367，均P〈0．01）。缺氧组细胞培养后12h和24h细胞中CD39和CD73蛋白的相对表达水平明显高于常氧组，差异均有统计学意义（均P〈0．05）。缺氧组细胞培Background Retinal hypoxia is one of primary causes of retinal neovascularization, and its mechanism is research hot topic. Studies showed that hypoxia stimulates the activation of many trancripation factors and vascular endothelial ceils, which leads to angiogenesis. Besides to the vascular endothelial growth factor, the effect of adenosine on angiogenesis is increasingly concerned. Objective This study was to invastigate the relationship of biological behaviour of human microvascular endothelial ceils （HMEC-1） under the hypoxia and adenosine ,and to explore the effect of adenosine on angiogenesis under hypoxia. Methods HMEC-1 cell line was cultured in vitro,and the ceils were divided into normoxia group and hypoxia group. The cells in the normoxia group were cultured under the 5% CO2 environment,and those in the hypoxia group were cultured under the 1% 02 ,94% N2 and 5% CO2 environment. The proliferation ability and percentage of the cells were assayed by cell counting kit-8 （CCK8） and EDU. The migration number and invasive number of the cells were detected by transwell chamber. The expressions of CD39 and CD73 proteins in the ceils were tested by Western blot and immunofluorescence technique,and the level of adenosine was measured by high performance liquid chromatography （HPLC）. Results The proliferation values （absobancy） were 0. 715±0. 067 and 0. 821±0. 056 in the normoxia group and the hypoxia group in 12 hours after culture, and those in 24 hours were 0. 946±0. 028 and 0. 998±0. 028 ,showing significant increase in the hypoxia group compared with the normoxia group （ t12h = 3. 805, t24h = 3. 222, all at P〈0. 01 ）. The precentage of the proliferation in the hypoxia group was evidently higher than that in the normoxia group （t =-6. 868, P〈O. 01 ）. The number of the cell migration and invasion in 24 hours after culture was 185.3±10. 594 and 74.2±10. 741 respectively in the normoxia group,and that in the hypoxia group was 300. 7±22. 853 and 107.5±7. 007, with significant di

关 键 词：新生血管 视网膜 微血管内皮细胞 人 血管生成 腺苷 

分 类 号：R774.1[医药卫生—眼科]